FULL LENGTH ORIGINAL PAPERS Ex~e~~e~t~ Laboratory Studies

pros.

s Bid PSychm NeuropsychopPrinted In Great Brttatn. All II&E resewed

1992, Vol. 16. pp. 943-967

0278 - 5846/92 $15.00 @ 1992 Pegamon

Press Ltd

NOVEL Ca2+ CURRENTS IN MAMMALIAN CNS NEURONS

NORIO AKAIKE, HISASHI YAMANAKA and MITSUTOSHI MUNAKATA Department of Neurophysiology, Tohoku University School of Medicine, Sendai, JAPAN

(Final form, January 1992)

Abstract Akaike, Norio, Hisashi Yamanaka and Mitsutoshi Munakata: Novel Ca*’ Currents in Mammalian CNS Neurons. Prog. Neuro-Psychopharmacol. & Biol. Psychiatry. 1992, 16(6): 943-957. 1.

2.

3. 4

5.

6.

Voltage-dependent Ca*+currents (I& in neurons can be classified into T-, N- and L-types. In the CA1 pyramidal neurons freshly dissociated from rat hippocampus we found an additional tetrodotoxin (‘ITX)-sensitive Ca” current (termed “lTX-1,‘). The TTX-1,. showed a heterogeneous distribution, preferentially in the dorsal site of CA1 region. Activation and inactivation processes of the TTX-I,. were highly potential-dependent, and the latter was fitted by a double exponential function. The ‘lTX-I, was activated at a threshold potential of about -55 mV and reached full activation at -30 mV. The steady-state inactivation of ‘RXI, could be fitted by a Boltxmann equation with a slope factor of 6.0 mV and a half-inactivation voltage of -72.5 mV. When the peak amplitudes of ‘ITX-Ica were plotted as a function of extracellular Ca*+concentration ([Ca*+]o),the current amplitude increased linearly without showing any saturation. The ratio of peak amplitude in the individual I-V relationships of Ca*‘, Srr’ and Ba*+ currents passing through the ‘RX-sensitive Ca*+-conducting channel was 1 : 0.33 : 0.05, although the current kinetics were much the same. ‘RX inhibited the ‘ITX-I, in time- and concentration-dependent manner without affecting the current kinetics. Lignocaine inhibited the TTX-I,* in a second in a concentration-dependent manner, with accelerating the inactivation process. The concentrations of half-inhibition (I&) were 3.5 x 10’ M for TTX and 3.6 x IO4 M for lignocaine. Scorpion toxin prolonged the inactivation phase of ?TX-Ica in a time- and concentration-dependent manner. In the toxin-treated neurons, both the slow time constant of inactivation (Q and its functional contribution to the total current increased with increasing the toxin concentration.

pevwor&: divalent cation. dorsal site, hippocampal CA1 region, dissociated pyramidal neuron, rat, scorpion toxin, tetrodotoxin-sensitive Ca current. Abbreviations: extracellular Ca*+concentration ([Ca*‘]o), extracellular Na’ concentration ([Na+lO),tetrodotoxin(lTX). 943

N.Akaikeet&

944

Introduction Pyramidal neurons in the CA1 subfield of the hippocampus are more sensitive to &hernia than those from other subfields (Alps et al 1987). When the density and distribution of damage induced by 4-min &hernia followed by a week of restitution were investigated in the rat hippocampus, only dorsal CA1 pyramidal neurons were found to be affected (Smith et al 1984). In hippocampal neurons, voltagedependent Ca” currents (I,..J were observed, which were classified into three groups (T-, N-, and Ltypes) on the basis of their kinetics, voltage-dependence, and unique pharmacologic sensitivities (Akaike et al 1989, Takahashi and Akaike 1991). Unexpectedly, we also found an additional ml-sensitive current (termed ‘FIX-I,)

Ca*’

in the pyramidal neurons of hippocampal CA1 dorsal site. In the present exper-

iment, therefore, we have investigated the regional distribution, the current kinetics, and the pharmacologic properties of the ‘FIX-I,

in the rat hippocampal CA1 region.

Methods

Pyramidal neurons in the hippocampal CA1 region were acutely dissociated from 2- to 3-week-old Wistar rats, as described previously (Akaike et al 1989). Briefly, SOO-pm thick hippocampal slices, obtained with a microslicer, were preincubated

in incubation solution (Takahashi et al 1989) saturated

with 95% 0, - 5% CO, for 50 min at room temperature (20 - 23 “C). Slices were then successively treated with 0.01% pronase for 15 - 20 min at 31 “C, and with 0.01 % thermolysin for 20 min at 31 “C. CA1 subfield of hippocampus was then micropunched out with a round tip of injection needle and transferred into a culture dish filled with well-oxygenated standard solution. CA1 pyramidal neurons were dissociated mechanically with a fire-polished micro-Pasteur pipette. Cells were used within 3 hr after the dissociation.

Na+- and K+-free external solution for recording ‘ITX-I,* had the following constitu-

ents (in mIvI): choline-Cl 140, CsCl5, CaCI, 10, HEPES 10, LaCl, 0.01 and glucose 10. The Na+- and K+-free internal solution had (in mM): N-methyl-Dglucamine

fluoride (NMG-F) 100, TEA-Cl 20 and

HEPES 10. Tris-base was used to adjust the pH of external and internal solutions to 7.4 and 7.2, respectively. TTX was dissolved in the external solution just before use. Voltage-clamp recording in the whole-cell configuration

was carried out at room temperature.

patch-clamp amplifier and monitored simultaneously

Ionic currents were recorded using a

after being filtered at 1 kI-Iz (NF Electronic In-

struments, FV-655) on a storage oscilloscope and a thermal-head pen-recorder.

Patch electrodes were

prepared on a vertical micropipette puller (Narishige, PB-7) and fire-polished on a microforge.

The

electrode resistance between a patch-pipette and the reference electrode was 3-6 MR. Net inward currents were measured after the transient capacitive and leak currents were subtracted. Leak currents were determined from hyperpolarizing

voltage steps equal in amplitude to those used to elicit the inward

currents. Measurements are given as the mean f standard error of the mean (S.E.M.).

TTX-sensitive

Ca2+

945

current

Student’s t test was carried out to assess statistical significance.

Results Hete oeeneous Distribution

of TTX-sensitive

Ca*+-conducting Channels in the Hippocampal CA1

I&R In Na+- and K+-free external and internal solutions, the L-type I,_. was observed in all the pyramidal neurons (n = 620) dissociated from CA1 subfield of rat hippocampus. whereas N-type was present only in 15 % of the neurons studied and was distributed without a specific regional preference. To separate the ‘ITX-sensitive and T-type. I, from L- and N-type I,, we perfused neurons, which did not exhibit Ntype I,, with F-containing internal solution to block the L-type Ca” channel (Akaike et al 1989). In CA1 pyramidal neurons (n = 32) dissociated from ventral hippocampus (No. 14 in Fig. 2C) the Ic, evoked by depolarizing the membrane to -20 mV from a holding potential (Vn) of -100 mV was blocked by 10m5M La%, an inorganic Ca2+channel blocker, but not with IO=]M ‘lTX, a concentration which was very effective in blocking voltage-dependent

Na+ current (IuJ (Kaneda et al 1989a)(Fig. 1A). This

suggests that the majority of the ventral CA1 neurons do not possess TTX-IcB. In contrast to those obtained from ventral hippocampus, the ICaevoked in CA1 pyramidal neurons dissociated from dorsal hippocampus (No. 4 in Fig. 2C) was not blocked by La3+ (n = 40). However, it was very sensitive to removal of extracellular Ca2’ and to a Na+ channel blocker. TI’X blocked this I, in a concentration- and time-dependent manner (Fig. 1B) without affecting its activation and inactivation kinetics. The halfmaximum inhibition concentration (IC& of ‘ITX was 3.5 nM. Since Ca*’ was the only pet-meant cation in this experimental condition, we concluded that the I-IX-sensitive voltage-dependent

Icll is carried by Ca’+ through the

Na+ channels, as is the case in squid giant axon (Meves and Vogel 1973), tunicate

egg cell (Okamoto et al 1976) and mammalian ventricular myocytes (Nilius 1988, Sorbera and Morad 1990). Our results are summarized in Fig. 2, where different colors show the heterogeneous distribution of ‘ITX- and T-type I, in the CA1 subfield of hippocampus. Figures 2A and 2B show coronal sections and schematic illustration of the rat hippocampus, respectively. Figure 2B was drawn by computer graphic reconstruction. The CA1 subfield of hippocampus was divided into 15 parts dorsolateraly (Fig. 20

In

30-50 neurons dissociated from each part (No. 1-15 in Fig. 2C), the extent of TTX- and T-type Icl contribution to the total inward current was determined electrophysiologically, based on their pharmacological and kinetic properties. Red indicates that 75-100 % of the total current depends on ‘ITX-IcB (82.43 f4.88 %, n = 108) and only O-25 % on T-type I,

Towards green the contribution of TTX-Ica decreases

as that of T-type I,. increases.

of ‘ITX-I,* in different color zones is: pink 62.05 +

The contribution

6.05 % (n = 115), yellow 30.63 f 7.54 % (n = 108) and green 16.81 f 3.91 % (n = 182). For T-type Ica: pink 25-50 %, yellow 50-75 % and green 75-100 %. The results show a predominant localization of ‘ITX- and T-type I, in the dorsal and ventral hippocampus, respectively.

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Fig. 1. Separation of ‘RX-I,, The pyramidal neurons were freshly dissociated from CA1 subfield of the hippocampus. The neurons were voltage-clamped in the whole-cell configuration. A rapid application of external solution was performed using concentration-clamp technique (Akaike et al 1986). Currents were activated by lOO- to 300-ms depolarizing pulses to -20 mV from a holding potential (V,) of -100 mV. A: T-type I,. recorded from a CA1 pyramidal neuron, dissociated from the ventral hippocampus (part No. 14 in Fig. 2C). Left, control; middle, in the presence of ‘ITX, right, in the presence of La3’. The T-type I, was abolished by addition of La 3+.B: I,* in a neuron isolated from the dorsal part (part No. 4 in Fig. 2C). Control (left). A slower inward current (T-type I,__,middle) was observed in the presence of ‘ITX. The faster inward current (‘ITX-I,, right) was not suppressed by La3+.

Current Kinetics of ‘lTX-I B The pyramidal neurons dissociated from the dorsal part of hippocampal CA1 region were perfused with Na+-free internal solution and with Nat-free external solution containing 10 mM Ca” and 10e5M La3+, at which concentration La3’ completely blocks all voltage-dependent ‘RX-I,.

was induced by a 60 ms step depolarization

current of ‘ITX-I,

T-, N- and L-type I,.. The

from a Vu of -100 mV. The maximum inward

appeared at about -30 mV in the current-voltage (I-V) relationship. The ‘ITX-I, did

not reverse even at depolarizing pulses over +lO mV (data not shown). The kinetic properties of ‘ITX-I,. were analyzed by measuring the activation time (time to peak) and the inactivation time (decay time constant), at different membrane potentials. The current decay of TI’XI, could be fitted by a sum of two components. The time to peak (t,) and the fast and slow decay time constants (T, and zi,, respectively) are plotted against the membrane potential (Fig. 3A, B). As can be seen, both the activation and inactivation of the TI’X-IcB are potential-dependent, the responses becoming more rapid with increasing depolarization. Figure 3A also shows the tr of both T-type I,. and INa plotted against the membrane potential. In this experiment, T-type IcII was recorded in neurons dissociated from the ventral part of the CA1 region. T-type Ica could be induced by a 300 ms step depolarization to various membrane potentials from a Vu of -100 mV in Na+-free external solution containing 10 mM-Ca*+ and 10e7M TTX. A semilogarithmic

plot of the inactivation

phase of I,& also showed a

double exponential decay whereas T-type ICadecayed as a single exponential function (Fig. 3B). rif and Zis of ‘ITX-IcB are about three times larger than those of INaat the potential range between -50 and -10

TTX-sensitive Caa* eurrent

III -6.72

I

It -%6

4.6

947

-3.6

A -256

Fig. 2, A: schematic ~llus~ation of heterogeueons d~s~b~tion of TTX-I,. and T-type Ica in coronal sections of rat hipp~ampus (see text), Arabic numerals show the distance from b~gma in mm scale. B: posterior view in compute-graphic 3-djmensional recons~ction of stippled area. The percent distribution of TTX- and T-type Ic. in the dorsal and ventral sites of rat hippoc~pus is given in text. Numbers I-II in Fig. 2A co~spond to those in Fig. 2C and indicate the sites where slices were obtained.

mV, but smaller than the time constant of T-type I,.

Both $ and zi of all three currents were strongly

po~nti~-dependenr.

Figure 3C shows the time courses of recovery (reactivation) from the com~Ie~e inactivation of TTXIa, &, and T-type I, a&a Vu of -100 mV, measured by a conve~~onal double pulse method (see inset). The recovery process of TTX-IcB as well as both Ina and T-type Ica could be fitted by a double exponential function. The faster component had a time constant of 13.1+ 1.0 ms (n = 4) and 84.3 f 4.2 % of recover

was dough

this process for TTX-Ic., 7.6 rt 1.1 ms (n = 4) and 89.6 5~1.4 % for I,, and 295 5~

67 ms (n = 6) and 79 rt 10 % for T-type I,, respectively. The slow component had a time constant of 12~2~

ms for TTX-I,., X0-180 ms for I,*, and 4-S s for T-type-I,, ~s~ctively.

The activation curve for TTX-I,

is shown in Fig. 4F, in which the threshold potential for T?“X-I,

activation was -55 mV. The full activation was achieved at -30 mV. The half-maximum

activation

~otenti~ was -42.5 mV, and the slope factor of 3 mV was obtained from a continuous curve fitted by Boltzmann equation. The steady-state inactivation (h ) curve of TTX-Ie. was obtains

using a conventional double pulse

method with 10 s prepulses to various potentials ranging from -120 to -40 mV (Fig. 4F). The relation-

TlX-senskive

Ca2+

current

-40

w

-20

w

(mv)

0

W)

C

w-+& 0

0

50

100

150

Interval(AT)

ZOO-

(ms)

(S)

Fig. 3. Comparison of the kinetics among ‘FIX-I,, Iua and T-type I,. in dissociated rat hippocampal pyramidal cells. Neurons were dissociated from the dorsal part for recording ‘ITX-IcI and from the ventral part for recording both I,. and T-type I,. The T-type Icl was measured in Na+-free external solution containing 10 mM-Ca’+ and 10‘7M TTX, at which concentration the TTX completely suppressed both the TIX-I,. and I,.. A: voltage dependence of activation time of TTX-I,.., (o), I,. (A) and T-type I,* (u). The activation time was measured as the time to peak ($). Mean values + S.E.M. from measurements on five neurons. In all cases V, was -100 mV. MP, membrane potential. B: voltage dependence of inactivation of T-TX-I,., Iua and T-type I,,. The inactivation rate was measured as the time constant of the exponential current decay (rJ. C: recovery from the complete inactivation of ‘ITXI, (0). I, (A) and T-type I,. @I).Testing depolarizing pulses to -20 mV were applied at different intervals (AT) after the end of a membrane depolarization of 100 ms for TTX-I,,, 50 ms for I, and 400 ms for T-type I& Vu was -100 mV in all cases. Each point is average value of four to five neurons.

ship between hopandprepulse potential was also fitted by Boltzmann equation. The ‘ITX-Ica was completely suppressed at a prepulse potential of -40 mV. The estimated V, s value was -72.5 mV with the prepulse duration of 10 s. The V,, value was little changed by further prolonging the prepulse beyond 10 s. When the duration of prepulse was shortened, the curves shifted toward the right (data not shown).

N.AkaikeetaL

950

In order to examine use-dependency, a train of command pulses (from a Vu of -100 mV to -20 mV) were. applied at different pulse intervals ranging from 1 to 50 Hz. All neurons were left in a resting state for more than 1 min before a train of stimulation.

Both ‘RX-I,. and I, remained fairly constant up to

~-HZ stimulation. When a frequency of stimulation was increased to more than 10 Hz, the inhibition of current amplitude appeared, and the inhibition cumulatively increased with each pulse. At 15-Hz stimulation the ‘ITX-I,* was considerably inhibited to about 20 % of the control, whereas the INawas inhibited to 70 %. With the increase in the rate of stimulation, the inhibition of current amplitude was stronger in the order of T-type I,. >> TTX-Ica > I,. TTX-IcB, INaand T-type Ice recovered completely from the inhibition after the cessation of a train of stimulation (data not shown). When 10 mM-Ca*+ in the external solution was replaced with Sr

or Ba*+, the inward currents pass-

ing through ‘FIX-sensitive Ca*+-conducting channels were reduced in amplitude (Fig. 4D). Also, the potential of maximum inward current in each I-V relationship was shifted slightly to the left, indicating the different effects of these divalent cations on the membrane surface charge ( Ohmori and Yoshii 1977). Therefore, the apparent activity sequence obtained from the maximum inward current through ‘FIX-sensitive Ca*+-conducting channels was in the order of Ca*+>> Srr’ >> Ba*+. The ratio of current amplitude was Ca *+ . Sra’ . Ba*’ = 1.00 : 0.33 : 0.05 (n = 6). As seen in Fig. 4E, a linear increase in TI’X-I,

was observed with increased extracellular Ca” concentration ([Ca*‘]J. The amplitude of IN1

also increased linearly without affecting the I-V relationship

as the extracellular Na+ concentration

([Na+lJ was increased. In contrast, the peak inward current of T-type IcDreached an almost complete saturation at around 40 mM [Ca2+10(24.4 mM Ca*+activity (“[Ca*+]J). Pharmacoloaical Properties of ‘FIX-I, Neurons were voltage-clamped at a Vu of -100 mV throughout the experiment. Depolarizing step of 80 mV lasting for 60 ms was applied at 1 Hz. No use-dependent change in the amplitude of TTX-I,_ was observed with this stimulus frequency. The inhibitory effect of TTX on TTX-I,, developed in a concentration- and time-dependent manner without affecting the activation and inactivation processes. Figure 4A summarizes the inhibition curve of TTX on the TTX-Ic.. ‘FIX depressing the TTX-I,

The threshold concentration

of

was less than 1U9 M. With regard to the action on the maximum ampli-

tude, a half-maximum inhibition concentration (IC& was 3.5 x 10m9M. The I - V relationship of ‘ITXI, did not shift in the presence of ITX (data not shown). The time course for reaching a steady-state of the inhibitory action shortened with increased ‘FIX concentration, but the time courses of recovery from the inhibition after washing out were quite the same at different TTX concentrations, indicating that the dissociation of ‘FIX from the binding site is independent of the ‘FIX concentration. Figure 4B shows the inhibitory action of lignocaine on TTX-Ica. The inhibition required only a few seconds to reach a steady-state inhibition at the concentrations used. There was no apparent difference between the time courses in the development of inhibition and recovery from the inhibition.

However,

the inhibitory action of lignocaine was associated with accelerated current kinetics of both activation and inactivation phases. Thereby, the time to peak and the half decay time of TI’X-IcB was shortened in

951

TIX-sensitive Ca2+ current

Time (ms)

~2’

pA

[Ca*+l,(mw

MP (mV)

Fig. 4. Electrical and pharmacological properties of ‘lTX-I,*. A: the current traces of Tl’X-1,. before and 0.5,1,2 and 3 min after the application of lo‘* M TTX. The dotted line in zero current level shows the holding current level (-100 mV). B: the ‘ITX-1c.s before and 1 and 10 set after application of lo” M lignocaine. C: effect of scorpion toxin on TlX-I,.. Superimposed currents (inset) recorded before and 3 min after the start of toxin application, and semilogarithmic plots of the inactivation phases of ‘ITX-1,&s with (right panel) or without (left panel) scorpion toxin (1 pg ml-‘). D: the I-V relationships for ‘RXsensitive IG, IsI and IBa. Vu was -100 mV. E: relationship between [CaZ’10and peak inward currents in the individual I - V relationships for TTX-I,.. All currents of TTX-I,* were normalized to the peak response recorded with 10 mM [Caz+lO.The ‘lTX-I, increased linearly with elevated [Ca”],. Each point represents the average.value from five neurons and the vertical bars indicate S.E.M.. F: activation and steady-state inactivation curves for “RX-I,.. Continuous curves of activation and steady-state inactivation were fitted by the Boltzmann equation using the least-square method. Inactivation was induced by 10 s prepulses just before testing depolarization to -20 mV.

the presence of this drug. The IC, was 3.6 x 10w4M. The scorpion toxin (1 pg ml-‘) prolonged the inactivation phase of TTX-IcI (Fig. 4C). The effect on the current inactivation was complete within 3 to 5 min after adding the toxin. The semilogarithmic plots of the inactivation phase of TlX-I,.

could be fitted with fast and slow exponential components (zif

and 2,. respectively) before (control) and 3 min after adding the toxin. When the amplitudes of fast and slow current components were estimated from the semilogarithmic plots (curve fitting) of the inactivation process of the ‘ITX-I,_, the scorpion toxin increased the fractional contribution

of slow current

component to the total current amplitude whereas the contribution of fast one decreased (Fig. 4C). The

N. Akahe

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etd.

,5mM

I

I 0

I 20

I 60

40

aPa+], W-W [Ca2+]o=5mM

(VH)

I

I

-25mV

I

I

, La3+=10%l r

-5mV

20ms

Fig. 5. Effect of [Na+l on TTX-I,*. All experiments were carried out in TTX-free external solution containing 10m5M-La”. A: relationship between the relative current amplitude and Na+ activity P[Na+l,). Note a linear increase in current amplitude as ‘[Na+], was increased. All currents were normalized to the peak inward current evoked by depolarizing pulse from a V, of -100 mV to -25 mV in B: Na*-free external solution ~n~n~g 5 mM-Ca a’. Each point is the average of four to six neurons, appearance of inward current having a ‘notch‘ in the inactivation phase by addition of extracellular Na+. Arrows show a ‘notch’, indicating that the inward current consists of fast and slow current components.

toxin had the same effect on Ina in this preparation (Kaneda et al 1989b). However, the scorpion toxin did not affect T-type I& As candidates for selective and potent blockers of T-type Ca” chmnel, the compounds such as amiloride, octanol and flunarizine were reported in mouse neuroblastoma (Tang et al 1988), guineapig brainstem slice (Llinas and Yarom 1986). chick sensory neuron (Fox et al 1987), and rat hypothalamic neuron (Akaike et al 1989). Therefore, these three compounds were tested on both TTX-1,. and T-type I,. which were evoked in 10 mM-Ca2+ external solution containing 1Q5 M-La3+ and I@’ M ‘RX, respectively. The neurons were held at a Vn of -100 mV and activated by a step depolarization to -30 mV at every 30 s with or without the blockers. Amiloride did not affect ‘ITX-I,B but suppressed the T-type I,,. The inhibitory effect of octanol was stronger on ‘RX-Ica (IC,, = 1.7 x 10m4M) than on T-type I,..&(IC, =

TIX-sensittve

953

Ca2+ current

2.8 x lOA M). Flunarizine suppressed equally the TTX-I,

(IC, = 1.2 x lo6 h4) and T-type ICr (IC,, =

1.0 x 1O-6Ivl). The kinetics of both currents were not affected by these drugs at concentrations used. When the effects of [Na+10on TTX-I,. evoked in external solution containing 5 mM-Ca’+ was examined, the inward current was enhanced linearly with increasing [Na+], from 0.1 to 60 mM, indicating that the TTX-I,* was not interfered by extracellular Na+ (Fig. 5A). In addition, at depolarizing step potentials beyond -25 mV, the inactivation phase of inward current in the external solution containing both 5 mM-Ca*+ and 10 mM-Na+ consisted of fast and slow current components (see arrows in Fig. 5B), which reflected the inactivation kinetics of voltage-dependent Na+ channels and TTX-sensitive Ca*+-conducting channels, respectively. Moreover, as seen in the lower panel of Fig. 5B, the amplitude of both the fast and slow current components increased with added Na+. The results suggest that Na+ is passing through not only the voltage-dependent

Na+ channels having fast kinetics but also the TTX-sensitive

Ca*+-conducting channels having somewhat slow kinetics.

We have demonstrated distinct voltage-dependence,

kinetics, ionic selectivity and pharmacological

characteristics of the ‘l-IX-sensitive Ca*+-conducting channels in the pyramidal neurones freshly dissociated from the rat hippocampal CA1 region. The kinetic properties of TTX-Ica were very similar to those of I,, and the TTX-I,. was unique in its high sensitivity to Na+ channel modulators such as TTX, lignocaine and scorpion toxin. The following results suggest that the TTX-I, Ca*+-conducting Na+ channels. (1) The transient TTX-I,

is passing through the

and INPwere activated at potentials above -60

mV and reached their current peaks at about -30 mV in a highly potential-dependent potentials of full activation were -30 mV for ITX-I, both TTX-I,

manner.

The

and -20 mV for INB.The inactivation processes of

and I, were fitted by a sum of two exponential functions. The midpotentials in the steady-

state inactivation curves of TI’X-I, and I, were -72.5 and -74.5 mV, respectively. (2) The ratio of time constant of the major component of recovery from the complete inactivation of TI’X-I,

and Iua was 1 :

0.6. (3) The order of frequency-dependent inhibition was ‘ITX-I,_, > I,,. (4) When the peak amplitudes in I - V relationship

of lTX-1,.

and Iua were plotted as a function of [Ca2’10and [NaClO,the current

amplitudes increased linearly with increasing [Ca2’10and [Na+lO,respectively. (5) The Na+ channel of hippocampal pyramidal neurones was highly sensitive to ‘lTX which suppressed the current in a concentration- and time-dependent fashion without affecting the current kinetics (Kaneda et al 1989a). They also

reported that lignocaine reduced IuII in a concentration-dependent

manner with accelerating the

current kinetics, indicating the blockade of open state of the Na+ channels (Yeh and Narahashi 1977). The IC, values of TTX and lignocaine on I, were 10-s M and 4 x 1P4 M, respectively. In the present experiment, the IC, values of ‘ITX and lignocaine on TTX-I,* were 3.5 x 10MgM and 3.6 x 1w4 M, respectively. was observed.

No difference in the inhibitory actions of TTX and lignocaine on both ‘ITX-I,. and INP The ‘ITX-IcI was insensitive to an inorganic Ca*’ blocker, La3+ of 10” M, at which

concentration T, N- and L-type Ca*’ channels were completely blocked (Takahashi et al 1989). (6) The

N.AkaikeetaL

954

action of scorpion toxin on the inactivation process of TTX-I,. was essentially similar to that of I,. in the frog node of Ranvier (Mozhayeva et al 1980; Benoit and Dubois 1987), squid axonal membrane (Narahashi et al 1972). and rat hippocampal pyramidal neuron (Kaneda et al 1989b). (7) The current amplitude of TTX-I,. was increased by adding Na+ extracellularly, indicating Na+ also can pass through this ‘ITX-sensitive Ca2+conducting channels. When the hippocampal pyramidal neurons were superfused with external solution containing 10 mMCa2+, St2’ or Ba2+,the ionic selectivity of the ‘ITX-sensitive Ca2+-conducting channels for these divalent cations was in the order of Ca” >> Srz’ > Ba ‘+. The selectivity differed markedly from the order (S? >> Ca2+> Ba2+) of T-type Ca2+channels in the same hippocampal preparation (Takahashi et al 1991), in rat hypothalamic neurons (Akaike et al 1989) and cultured hippocampal neuron from fetal rat (Yaari et al 1987). A similar ionic selectivity, Ca” >> Sr” > Ba2+ = 0, was observed for the ‘Ca2+ current’ through ‘Na+ channels’ having faster current kinetics than the ‘Ca’+ current’ through ‘Ca” channels’ in egg cell membrane of the tunicate (Okamoto et al 1976). In CA1 pyramidal neurons of rat hippocampus, immunocytochemical

studies localized TTX- and

saxitoxin-sensitive Rr and R,, subtypes of Na’ channels in cell body and fiber layers (Gordon et al 1987, Westenbroek et al 1989). When the R,, subtype of Na+ channel was expressed in Xenopus oocytes it mediated rapidly activating and completely inactivating INI (Goldin et al 1986, Noda et al 1986). Since as yet the expression of R, subtype in Xenopus oocytes was not successful, its physiological properties are not known (Noda et al 1986). Nevertheless, in hippocampal pyramidal cell body, the R, Na’ channel subtype may be responsible for TTX-sensitive slow inward current (‘ITX-IcJ that lasted tens of milliseconds before being inactivated (French and Gage 1985). In CNS neurons the T-type Ca2+channels play an important role in functions by contributing to spontaneous depolarization waves as a generator of pacemaker activity and a rebound excitation (Llinas and Yarom 1981, Choi 1988).

The ‘ITX-I,. and T-type Ica were heterogeneously distributed in the dorsal

and ventral sites of rat hippocampal CA1 region, and the amplitude of ‘lTX-IcI1 in the dorsal portion was almost equal to that of T-type ICa in the ventral portion (Akaike et al 1991). Both the currents were activated at around -60 mV near the resting potential.

In addition, the half-recovery

time from the

complete inactivation of TTX-Icl was about 20 to 30 times more rapid than that of T-type I,,. Conceming the possible role of the ‘ITX-I, in the hippocampal pyramidal neurons, therefore, the results suggest that the TTX-1,. may act like I,. and contribute more effectively to spontaneous depolarization waves and bursting activities of the pyramidal neurons than does T-type I,.. The hippocampal CA1 pyramidal cells are highly sensitive to ischemia among brain neurons (Blomquist and Wieloch 1985, Alps et al 1986,1987). Interestingly, an anticonvulsant agent, phenytoin, significantly reduced the ischemic brain damage in animals after the occlusion of arteries (Cullen et al 1979, Artru and Michenfelder 1980). The phenytoin also blocked I,* in isolated rat hippocampal pyramidal neurons of CA1 region in the order of TTX-I,* > T-type I,. >> N-type I,. > L-type I,. (Takahashi et al 1989). These results suggest a significant linkage between hypoxic-ischemic neuronal damage and the lethal Ca2+influxes through various Caz+-conducting channels. Moreover, when the density and disnibu-

TIX-sensitive

Ca2+

955

current

tion of hlppocampal damage after 4 min of &hernia, followed by 5 days of restitution, was studied in the rat, the cell death was observed only in the CA1 region. Furthermore, much damage was found not in the ventral site of the CA1 region but in the dorsal site (Smith et al 1984) where the TI’X-sensitive Ca*+conducting channels exist. In fact, when TTX at a concentration of IO-’ to 10m6M was topically applied to the rat hippocampal CA1 subfield before 30 min of ischemia, the ‘ITX mitigated the ischemic hippocampal neuronal damage in limited but concentration-dependent by, the excessive activation of TTX-sensitive Ca”-conducting

manner (Yamasaki et al 1991). Therechannels might be significantly linked to

the Ca*+-mediated nemotoxicities in CNS neurons,

Conclusion It was concluded that the TTX-Ic, is carried by Ca*’ but through the Na+ channels. The ‘ITX-I, may create much effective mechanism for generation of membrane potential oscillation and autorhythmicity as compared with T-type Ica, suggesting that the excess excitation of ‘ITX-sensitive Ca”-conducting channels might be associated with the cell death in the dorsal portion of CA1 region where the channels exist.

AcknowledPement This study was supported by Grants-in-Aid for Scientific Research (Nos. 2557005,03304026)

to N.

Akaike from The Ministry of Education, Science and Culture, JAPAN.

AKAIKE, N., INOUE, M. and KRISHTAL, 0. A. (1986) ‘Concentration-Clamp’ Study of ‘IAminobutyric Acid-Induced Chloride Current Kinetics in Frog Sensory Neurones. I. Physiol. m: 171-185. AKAIKE, N., KOSTYUK, P. G. and OSIPCHUK, Y. V. (1989) Dihydropyridine-Sensitive LowThreshold Calcium Channels in Isolated Rat Hypothalamic Neurones. J. Physiol. 412: 181-195. AKAIKE, N., TAKAHASHI, K. and MORIMOTO, M. (1991) Heterogeneous Distribution of Tetrodotoxin-Sensitive Calcium-Conducting Channels in Rat Hippocampal CA1 Neurons. Brain Res. m: 135-138. ALPS, B. J., CALDER, C., and WILSON, A. D. (1986) The Effect of Nicardipine on “Delayed Neuronal Death” in the Ischaemic Gerbil Hippocampus. Br. J. Pharmacol. 88: 250P. ALPS. B. J., CALDER, C., HAAS, W. K. and WILSON, A. D. (1987) The Delayed Post-Ischaemic Treatment Effects of Nicardipine in a Rat Model of Four Vessel Occlusion. Br. J. Pharmacol. a: 312P. ARTRU, A. A. and MICHENFELDER, J. I?. (1980) Cerebral Protective, Metabolic and Vascular Effect of Phenytoin. Stroke 2: 337-382. BENOIT, E. and DUBOIS, J. (1987) Properties of maintained Sodium Current Induced by a Toxin from Androctonw Scorpion in Frog Node of Ranvier. J. Physiol. m 93-l 14. BLOMQUIST, P. and WIELOCH, T. (1985) Ischemic Brain Damage in the Rat Following Cardiac

956

N. Akaike et al.

Arrest Using a Long-Term Recovery Model. J. Cereb. Blood Flow Metab. a 420-432. CHOI, D.W. (1988) Calcium-Mediated Neurotoxlcity: Relationship to Specific Channel Types and Role in Ischemic Damage. Trends Neurosci. 1L: 465469. CULLEN, J. P., ALDRETE, J. A., JANKOVSKY, L. and ROMO-SALAS, F. (1979) Protective Action of Phenytoin in Cerebral Ischemia. Anesth. Anal. Ss: 165169. FOX A. P., NOWYCKY, M. C. and TSIEN, R. (1987) Kinetic and Pharmacological Properties Distinuishmg Three Types of Calcium Currents in Chick Sensory Neurones. J. Physiol. B: 149-172. FRENCH, C. R. and GAGE, P. W. (1985) A Threshold Sodium Current in Pyramidal Cells in Rat Hippocampus. Neurosci. Lett. s: 289-294. GOLDIN, A. L., SNUTCH. T., LUBBERT. H., DOWSETT, A., MARSHALL, J., AULD, V., DOWNEY, W., FRITZ, L. C., LESTER, H. A., DUNN, R., CA’ITERALL, W. A. and DAVIDSON, N. (1986) Messenger RNA Coding for Only the a Subunit of the Rat Brain Na Channel is Sufficient for Expression of Functional Channels in Xenopus Oocytes. Proc. Natl. Acad. Sci. USA. u: 75037507. GORDON, D., MERRICK, D., AULD, V., DUNN, R., GOLDIN, A. L., DAVIDSON, N. and CATTERALL, W. A. (1987) Tissue-Specific Expression of the R, and R,, Sodium Channel Subtypes. Proc. Natl. Acad. Sci. USA. && 8682-8686. KANEDA, M., OYAMA. Y., IKEMOTO, Y. and AKAIKE, N. (1989a) Blockade of the VoltageDependent Sodium Current in Isolated Rat Hippocampal Neurons by Tetrodotoxin and Lidocaine. Brain Res. & 348-351. KANEDA, M., OYAMA, Y., IKEMOTO, Y. and AKAIKE, N. (1989b) Scorpion Toxin Prolongs an Inactivation Phase of the Voltage-Dependent Sodium Current in Rat Isolated Single Hippocampal Neurons. Brain Res. m 192- 195. LLINAS, R. and YAROM, Y. (1981) Electrophysiology of Mammalian Inferior Olivary Neurones in Vitro. Different Types of Voltage-Dependent Ionic Conductances. J. Physiol. m 549-567. LLINAS. R. and YAROM, Y. (1986) Specific Blockade of the Low Threshold Calcium Channel by High Molecular Weight Alcohols. Sot. Neurosci. Abstr. 174. MEVES, H. and VOGEL, W. (1973) Calcium Inward Currents in Internally Perfused Giant Axons. J. Physiol. m: 225-265. MOZHAYEVA, G. N., NAUMOV, A. P., NOSYREVA, E. D. and GRISHIN, E. V. (1980) PotentialDependent Interaction of Toxin Venom of the Scorpion &thus Eupeus with Sodium Channels in Myelinated Fibre. Biochem. Biophys. Acta m: 587602. NARAHASHI, T., SHAPIRO, B. I., DEGUCHI, T., SCUKA, M. and WANG, C. M. (1972) Effects of Scorpion Venom on Squid Axon Membranes. Am. J. Physiol. 222: 850-857. NILIUS. B. (1988) Calcium Block of Guinea-Pig Heart Sodium Channels with and without Modification by the Piperaxinylmdole DPI 201-106, J. Physiol. m: 537. NODA, M, IKEDA, T., SUZUKI, H., TAKESHIMA, H., TAKAHASHI, T., KUNO, M., and NUMA, S. (1986) Expression of Functional Sodium Channels from Cloned cDNA. Nature 322: 826-828. OHMORI, H. and YOSHII, M. (1977) Surface Potential Reflected in Both Gating and Permeation Mechanisms of Sodium and Calcium Channels of the Tunicate Egg Cell Membrane. J. Physiol. m 429-463. OKAMOTO, H., TAKAHASHI, K. and YOSHII, M. (1976) Two Components of the Calcium Current in the Egg Cell Membrane of the Tunicate. J. Physiol, 255: 527-561. SMITH, M. L., AUER, R. N. and SIESJO, B. K. (1984) The Density and Distribution of Ischemic Brain Injury in the Rat Following 2-10 Min of Forebrain Ischemia. Acta Neuropathol. @: 319-332. SORBERA, L. A. and MORAD, M. (1990) Atrionatriuretic Peptide Transforms Cardiac Sodium Channels into Calcium-Conducting Channels. Science m 969-973. TAKAHASHI, K. and AKAIKE, N. (1991) Calcium Antagonist Effects on Low-Threshold (T-Type) Calcium Current in Rat Isolated Hippocampal CA1 Pyramidal Neurons. J. Pharm. Exp. Therap. a: 169-175.

1TX-sensitive Ca2+ current

957

TAKAHASHI, K.. WAKAMORI, M. and AKAIKE, N. (1989) Hippocampal CA1 Pyramidal Cells of Rats Have Four Voltage-Dependent Calcium Conductances. Neurosci. Lett. && 229-234. TANG, C. M., PRESSER, F. and MORAD, M. (1988) AmiIoride Selectively Blocks the Low Threshold (T) Calcium Channel. Scinece m: 213-215 WESTENBROEK, R. E., MERRICK, D. K. and CA’ITERALL, W. A. (1989) Differential Subcellular Localization of the R, and Ru Na+ Channel Subtypes in Central Neurons. Neuron 9: 695-704. YAMASAKI, Y.. KOGURE, K., HARA, H.. BAN, H. and AKAIKE, N. (1991) The Possible Involvement of Tetrodotoxin-Sensitive Ion Channels in Ischemic Neuronal Damage in the Rat Hippocampus. Neurosci. Lett. 121: 251-254. YAARI, Y., HAMON, B. and LUX H. D. (1987) Development of Two Types of Calcium Channels in Cultured Mammalian Hippocampal Neurons. Science 225: 680-682. YEH, J. Z. and NARAHASHI, T. (1977) Kinetic Analysis of Pancuronium Interaction with Sodium Channels in Squid Axon Membranes. J. Gen. Physiol. 6p: 293-323.

Inquiries and reprint requests should be addressed to: Prof. Norio Akaike Department of Neurophysiology Tohoku University School of Medicine 1- 1 Seiryo-cho. Aoba-ku Sendai, 980 JAPAN